Small cell lung carcinoma (SCLC) is the most aggressive form of lung cancer. With dismal survival rates and a lack of targeted therapies it is critical that we understand the major genes and pathways that drive SCLC. To identify new potential SCLC drivers, we have sequenced human SCLC exomes and transcriptomes and found frequent inactivating mutations in the histone methyltransferase MLL2. The Cancer Genome Atlas study of squamous lung carcinoma (SQCC) revealed that MLL2 is among the most frequently mutated genes in SQCC, suggesting that MLL2 may be a tumor suppressor across multiple lung cancer types. MLL2 is frequently mutated across many human cancers but there has been little study of the link between MLL2 and cancer. Our preliminary data provide functional evidence that MLL2 is a tumor suppressor. By conferring histone H3K4 monomethylation, MLL2 is newly emerging as a key regulator of transcriptional enhancer function but this role has not been explored in tumorigenesis. In this grant proposal, we interrogate MLL2 as a critical tumor suppressor in lung cancer. Specific Aim 1: To test the hypothesis that MLL2 is a tumor suppressor in lung cancer. We have previously used a sensitized autochthonous mouse model of SCLC to show that an SCLC-mutated gene is functionally important for SCLC. We propose to generate new models of MLL2-mutant SCLC and SQCC. The models will not only reveal mechanisms underlying MLL2 tumor suppressor activity but will be ideal preclinical tools for future testing of novel therapies directed towards vulnerabilities conferred by MLL2 inactivation. Specific Aim 2: To identify target genes and pathways regulated by MLL2 that promotes cancer. MLL2 is a methyltransferase that regulates histone H3K4 monomethylation, a mark of transcriptional enhancers. We hypothesize that MLL2 regulation of transcriptional enhancers controls the expression of tumor suppressive transcripts important for lung cancer suppression. We will use primary tumors and cell culture studies to identify transcriptional changes that depend on MLL2 status. Pathway analyses will determine whether known signaling pathways important for SCLC are altered upon MLL2 perturbation and will identify new pathways that may be important for SCLC. To identify direct MLL2-regulated enhancers in SCLC, ChIPseq analyses will determine which of the differentially expressed genes exhibit direct MLL2 binding and, upon MLL2 inactivation, decreased histone H3K4 monomethylation and other marks associated with active enhancers. A subset of direct MLL2 target genes may act themselves as SCLC tumor suppressors and will be examined for functional roles in SCLC. These studies will elucidate molecular underpinnings of MLL2-mutant lung cancer and will provide new mouse models for preclinical studies. As there are no targeted therapies for SCLC or SQCC, it is critical that we use rigorous mouse models to dissect the role of MLL2 in lung cancer.